Method for linear mRNA amplification

ABSTRACT

Methods for linearly amplifying mRNA to produce antisense RNA are provided. In the subject methods, mRNA is converted to double-stranded cDNA using a promoter-primer having a poly-dT primer site linked to a promoter sequence so that the resulting double-stranded cDNA is recognized by an RNA polymerase. The resultant double-stranded cDNA is then transcribed into antisense RNA in the presence of a reverse transcriptase that is rendered incapable of RNA-dependent DNA polymerase activity during this transcription step. The subject methods find use a variety of different applications in which the preparation of linearly amplified amounts of antisense RNA is desired. Also provided are kits for practicing the subject methods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of copending U.S. patent applicationSer. No. 09/690,173, filed Oct. 16, 2000, which is a continuation ofU.S. patent application Ser. No. 09/322,692, filed May 28, 1999, nowissued U.S. Pat. No. 6,132,997 from which priority is claimed under 35U.S.C. 120. The entireties of these applications are incorporated hereinby reference.

INTRODUCTION

1. Technical Field

The technical field of this invention is the enzymatic amplification ofnucleic acids.

2. Background of the Invention

The characterization of cellular gene expression finds application in avariety of disciplines, such as in the analysis of differentialexpression between different tissue types, different stages of cellulargrowth or between normal and diseased states. Fundamental todifferential expression analysis is the detection of different mRNAspecies in a test population, and the quantitative determination ofdifferent mRNA levels in that test population. However, the detection ofrare mRNA species is often complicated by one or more of the followingfactors: cell heterogeneity, paucity of material, or the limits ofdetection of the assay method. Thus, methods which amplify heterogeneouspopulations of mRNA that do not introduce significant changes in therelative amounts of different mRNA species facilitate this technology.

A number of methods for the amplification of nucleic acids have beendescribed. Such methods include the “polymerase chain reaction” (PCR)(Mullis et al., U.S. Pat. No. 4,683,195), and a number oftranscription-based amplification methods (Malek et al., U.S. Pat. No.5,130,238; Kacian and Fultz, U.S. Pat. No. 5,399,491; Burg et al., U.S.Pat. No. 5,437,990). Each of these methods uses primer-dependent nucleicacid synthesis to generate a DNA or RNA product, which serves as atemplate for subsequent rounds of primer-dependent nucleic acidsynthesis. Each process uses (at least) two primer sequencescomplementary to different strands of a desired nucleic acid sequenceand results in an exponential increase in the number of copies of thetarget sequence. These amplification methods can provide enormousamplification (up to billion-fold). However, these methods havelimitations which make them not amenable for gene expression monitoringapplications. First, each process results in the specific amplificationof only the sequences that are bounded by the primer binding sites.Second, exponential amplification can introduce significant changes inthe relative amounts of specific target species—small differences in theyields of specific products (for example, due to differences in primerbinding efficiencies or enzyme processivity) become amplified with everysubsequent round of synthesis.

Amplification methods that utilize a single primer are amenable to theamplification of heterogeneous mRNA populations. The vast majority ofmRNAs carry a homopolymer of 20-250 adenosine residues on their 3′ ends(the poly-A tail), and the use of poly-dT primers for cDNA synthesis isa fundamental tool of molecular biology. “Single-primer amplification”protocols have been reported (see e.g. Kacian et al., U.S. Pat. No.5,554,516; Van Gelder et al., U.S. Pat. No. 5,716,785). The methodsreported in these patents utilize a single primer containing an RNApolymerase promoter sequence and a sequence complementary to the 3′-endof the desired nucleic acid target sequence(s) (“promoter-primer”). Inboth methods, the promoter-primer is added under conditions where ithybridizes to the target sequence(s) and is converted to a substrate forRNA polymerase. In both methods, the substrate intermediate isrecognized by RNA polymerase, which produces multiple copies of RNAcomplementary to the target sequence(s) (“antisense RNA”). Each methoduses, or could be adapted to use, a primer containing poly-dT foramplification of heterogeneous mRNA populations.

Amplification methods that proceed linearly during the course of theamplification reaction are less likely to introduce bias in the relativelevels of different mRNAs than those that proceed exponentially. In themethod described in U.S. Pat. No. 5,554,516, the amplification reactioncontains a nucleic acid target sequence, a promoter-primer, an RNApolymerase, a reverse transcriptase, and reagent and buffer conditionssufficient to allow amplification. The amplification proceeds in asingle tube under conditions of constant temperature and ionic strength.Under these conditions, the antisense RNA products of the reaction canserve as substrates for further amplification by non-specific primingand extension by the RNA-dependent DNA polymerase activity of reversetranscriptase. As such, the amplification described in U.S. Pat. No.5,554,516 proceeds exponentially. In contrast, in specific examplesdescribed in U.S. Pat. No. 5,716,785, cDNA synthesis and transcriptionoccur in separation reactions separated by phenol/chloroform extractionand ethanol precipitation (or dialysis), which may incidentally allowfor the amplification to proceed linearly since the RNA products cannotserve as substrates for further amplification.

The method described in U.S. Pat. No. 5,716,785 has been used to amplifycellular mRNA for gene expression expression monitoring (for example, R.N. Van Gelder et al. (1990), Proc. Natl. Acad. Sci. USA 87, 1663; D. J.Lockhart et al. (1996), Nature Biotechnol. 14, 1675). However, thisprocedure is not readily amenable to high throughput processing. Inpreferred embodiments of the method described in U.S. Pat. No.5,716,785, poly-A mRNA is primed with a promoter-primer containingpoly-dT and converted into double-stranded cDNA using a method describedby Gubler and Hoffman (U. Gubler and B. J. Hoffman (1983), Gene 25,263-269) and popularized by commercially available kits for cDNAsynthesis. Using this method for cDNA synthesis, first strand synthesisis performed using reverse transcriptase and second strand cDNA issynthesized using RNaseH and DNA polymerase I. After phenol/chloroformextraction and dialysis, double-stranded cDNA is transcribed by RNApolymerase to yield antisense RNA product. The phenol/chloroformextractions and buffer exchanges required in this procedure are laborintensive, and are not readily amenable to robotic handling.

Accordingly, there is interest in the development of improved methods ofantisense RNA amplification. Of particular interest would be thedevelopment of a linear amplification protocol that did not require areverse transcriptase separation step.

Relevant Literature

United States Patents disclosing methods of antisense RNA synthesisinclude: U.S. Pat. Nos. 5,716,785; 5,554,516; 5,545,522; 5,437,990;5,130,238; and 5,514,545. Antisense RNA synthesis is also discussed inPhillips and Eberwine (1996), Methods: A companion to Methods inEnzymol. 10, 283; Eberwine et al. (1992), Proc., Natl., Acad. Sci. USA89, 3010; Eberwine (1996), Biotechniques 20, 584; and Eberwine et al.(1992), Methods in Enzymol. 216, 80.

SUMMARY OF THE INVENTION

Methods for linearly amplifying mRNA to produce RNA (particularly asantisense RNA) are provided. In the subject methods, mRNA is convertedto cDNA (particularly double-stranded cDNA) using a promoter-primerparticularly having a poly-dT primer site linked to a promoter sequenceso that the resulting cDNA is recognized by an RNA polymerase. Theresultant cDNA is then transcribed into RNA (particularly antisense RNA)in the presence of a reverse transcriptase that is rendered incapable ofRNA-dependent DNA polymerase activity during this transcription step.The subject methods find use a variety of different applications inwhich the preparation of linearly amplified amounts of antisense RNA isdesired. Also provided are kits for practicing the subject methods.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 provides a graphical representation of the results obtained usingthe subject methods versus those described in U.S. Pat. No. 5,716,785.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Methods for linearly amplifying mRNA to produce antisense RNA areprovided. In the subject methods, mRNA is converted to double-strandedcDNA using a promoter-primer having a poly-dT primer site linked to apromoter sequence so that the resulting double-stranded cDNA isrecognized by an RNA polymerase. The resultant double-stranded cDNA isthen transcribed into antisense RNA in the presence of a reversetranscriptase that is rendered incapable of RNA-dependent DNA polymeraseactivity during this transcription step. The subject methods find use ina variety of different applications in which the preparation of linearlyamplified amounts of antisense RNA is desired. Also provided are kitsfor practicing the subject methods.

Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

It must be noted that as used in this specification and the appendedclaims, the singular forms “a”, an” and “the” include plural referenceunless the context clearly dictates otherwise. Unless defined otherwiseall technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs.

The subject invention provides methods for linearly amplifying mRNA intoantisense RNA. As such, the subject invention provides methods ofproducing amplified amounts of antisense RNA from an initial amount ofmRNA. By amplified amounts is meant that for each initial mRNA, multiplecorresponding antisense RNAs, where the term antisense RNA is definedhere as ribonucleic acid complementary to the initial mRNA, areproduced. By corresponding is meant that the antisense RNA shares asubstantial amount of sequence identity with the sequence complementaryto the mRNA (i.e. the complement of the initial mRNA), where substantialamount means at least 95% usually at least 98% and more usually at least99%, where sequence identity is determined using the BLAST algorithm, asdescribed in Altschul et al. (1990), J. Mol. Biol. 215:403-410 (usingthe published default setting, i.e. parameters w=4, t=17). Generally,the number of corresponding antisense RNA molecules produced for eachinitial mRNA during the subject linear amplification methods will be atleast about 10, usually at least about 50 and more usually at leastabout 100, where the number may be as great as 600 or greater, but oftendoes not exceed about 1000.

In the first step of the subject methods, an initial mRNA sample issubjected to a series of enzymatic reactions under conditions sufficientto ultimately produce double-stranded DNA for each initial mRNA in thesample that is amplified. During this first step, an RNA polymerasepromoter region is incorporated into the resultant product, which regionis critical for the second step of the subject methods, i.e. thetranscription step described in greater detail infra.

The initial mRNA may be present in a variety of different samples, wherethe sample will typically be derived from a physiological source. Thephysiological source may be derived from a variety of eukaryoticsources, with physiological sources of interest including sourcesderived from single-celled organisms such as yeast and multicellularorganisms, including plants and animals, particularly mammals, where thephysiological sources from multicellular organisms may be derived fromparticular organs or tissues of the multicellular organism, or fromisolated cells derived therefrom. In obtaining the sample of RNA to beanalyzed from the physiological source from which it is derived, thephysiological source may be subjected to a number of differentprocessing steps, where such processing steps might include tissuehomogenization, cell isolation and cytoplasm extraction, nucleic acidextraction and the like, where such processing steps are known to thoseof skill in the art. Methods of isolating RNA from cells, tissues,organs or whole organisms are known to those of skill in the art and aredescribed in Maniatis et al. (1989), Molecular Cloning: A LaboratoryManual 2d Ed. (Cold Spring Harbor Press). Alternatively, at least someof the initial steps of the subject methods may be performed in situ, asdescribed in U.S. Pat. No. 5,514,545, the disclosure of which is hereinincorporated by reference.

Depending on the nature of the primer employed during first strandsynthesis, as described in greater detail below, the subject methods canbe used to produce amplified amounts of antisense RNA corresponding tosubstantially all of the mRNA present in the initial sample, or to aproportion or fraction of the total number of distinct mRNAs present inthe initial sample. By substantially all of the mRNA present in thesample is meant more than 90%, usually more than 95%, where that portionnot amplified is solely the result of inefficiencies of the reaction andnot intentionally excluded from amplification.

The promoter-primer employed in the amplification reaction includes: (a)a poly-dT region for hybridization to the poly-A tail of the mRNA; and(b) an RNA polymerase promoter region 5′ of the -poly-dT region that isin an orientation capable of directing transcription of antisense RNA.In certain embodiments, the primer will be a “lock-dock” primer, inwhich immediately 3′ of the poly-dT region is either a “G”, “C”, or “A”such that the primer has the configuration of 3′-XTTTTTTTT . . . 5′,where X is “G”, “C”, or “A”. The poly-dT region is sufficiently long toprovide for efficient hybridization to the poly-A tail, where the regiontypically ranges in length from 10-50 nucleotides in length, usually10-25 nucleotides in length, and more usually from 14 to 20 nucleotidesin length.

A number of RNA polymerase promoters may be used for the promoter regionof the first strand cDNA primer, i.e. the promoter-primer. Suitablepromoter regions will be capable of initiating transcription from anoperationally linked DNA sequence in the presence of ribonucleotides andan RNA polymerase under suitable conditions. The promoter will be linkedin an orientation to permit transcription of antisense RNA. A linkeroligonucleotide between the promoter and the DNA may be present, and if,present, will typically comprise between about 5 and 20 bases, but maybe smaller or larger as desired. The promoter region will usuallycomprise between about 15 and 250 nucleotides, preferably between about17 and 60 nucleotides, from a naturally occurring RNA polymerasepromoter or a consensus promoter region, as described in Alberts et al.(1989) in Molecular Biology of the Cell, 2d Ed. (Garland Publishing,Inc.). In general, prokaryotic promoters are preferred over eukaryoticpromoters, and phage or virus promoters most preferred. As used herein,the term “operably linked” refers to a functional linkage between theaffecting sequence (typically a promoter) and the controlled sequence(the mRNA binding site). The promoter regions that find use are regionswhere RNA polymerase binds tightly to the DNA and contain the start siteand signal for RNA synthesis to begin. A wide variety of promoters areknown and many are very well characterized. Representation promoterregions of particular interest include T7, T3 and SP6 as described inChamberlin and Ryan, The Enzymes (ed. P. Boyer, Academic Press, NewYork) (1982) pp 87-108.

Where one wishes to amplify only a portion of the mRNA species in thesample, one may optionally provide for a short arbitrary sequence 3′ ofthe poly- dT region, where the short arbitrary sequence will generallybe less than 5 nucleotides in length and usually less than 2 nucleotidesin length, where the dNTP immediately adjacent to the poly-dT regionwill not be a T residue and usually the sequence will comprise no Tresidue. Such short 3′ arbitrary sequences are described in Ling andPardee (1992), Science 257, 967.

The promoter-primer described above and throughout this specificationmay be prepared using any suitable method, such as, for example, theknown phosphotriester and phosphite triester methods, or automatedembodiments thereof. In one such automated embodiment, dialkylphosphoramidites are used as starting materials and may be synthesizedas described by Beaucage et al. (1981), Tetrahedron Letters 22, 1859.One method for synthesizing oligonucleotides on a modified solid supportis described in U.S. Pat. No. 4,458,066. It is also possible to use aprimer that has been isolated from a biological source (such as arestriction endonuclease digest). The primers herein are selected to be“substantially” complementary to each specific sequence to be amplified,i.e.; the primers should be sufficiently complementary to hybridize totheir respective targets. Therefore, the primer sequence need notreflect the exact sequence of the target, and can, in fact be“degenerate.” Non-complementary bases or longer sequences can beinterspersed into the primer, provided that the primer sequence hassufficient complementarity with the sequence of the target to beamplified to permit hybridization and extension.

In the first step of the subject method, the oligonucleotidepromoter-primer is hybridized with an initial mRNA sample and theprimer-mRNA hybrid is converted to a double-stranded cDNA product thatis recognized by an RNA polymerase. The promoter-primer is contactedwith the mRNA under conditions that allow the poly-dT site to hybridizeto the poly-A tail present on most mRNA species. The catalyticactivities required to convert primer-mRNA hybrid to double-strandedcDNA are an RNA-dependent DNA polymerase activity, a RNaseH activity,and a DNA-dependent DNA polymerase activity. Most reversetranscriptases, including those derived from Moloney murine leukemiavirus (MMLV-RT), avian myeloblastosis virus (AMV-RT), bovine leukemiavirus (BLV-RT), Rous sarcoma virus (RSV) and human immunodeficiencyvirus (HIV-RT) catalyze each of these activities. These reversetranscriptases are sufficient to convert primer-mRNA hybrid todouble-stranded DNA in the presence of additional reagents whichinclude, but are not limited to: dNTPs; monovalent and divalent cations,e.g. KCl, MgCl₂; sulfhydryl reagents, e.g. dithiothreitol; and bufferingagents, e.g. Tris-Cl. Alternatively, a variety of proteins that catalyzeone or two of these activities can be added to the cDNA synthesisreaction. For example, MMLV reverse transcriptase lacking RNaseHactivity (described in U.S. Pat. No. 5,405,776) which catalyzesRNA-dependent DNA polymerase activity and DNA-dependent DNA polymeraseactivity, can be added with a source of RNaseH activity, such as theRNaseH purified from cellular sources, including Escherichia coli. Theseproteins may be added together during a single reaction step, or addedsequentially during two or more substeps. Finally, additional proteinsthat may enhance the yield of double-stranded DNA products may also beadded to the cDNA synthesis reaction. These proteins include a varietyof DNA polymerases (such as those derived from E. coli, thermophilicbacteria, archaebacteria, phage, yeasts, Neurosporas, Drosophilas,primates and rodents), and DNA Ligases (such as those derived from phageor cellular sources, including T4 DNA Ligase and E. coli DNA Ligase).

Conversion of primer-mRNA hybrid to double-stranded cDNA by reversetranscriptase proceeds through an RNA:DNA intermediate which is formedby extension of the hybridized promoter-primer by the RNA-dependent DNApolymerase activity of reverse transcriptase. The RNaseH activity of thereverse transcriptase then hydrolyzes at least a portion of the RNA:DNAhybrid, leaving behind RNA fragments that can serve as primers forsecond strand synthesis (Meyers et al., Proc. Nat'l Acad. Sci. USA(1980) 77:1316 and Olsen & Watson, Biochem. Biophys. Res. Comm. (1980)97:1376). Extension of these primers by the DNA-dependent DNA polymeraseactivity of reverse transcriptase results in the synthesis ofdouble-stranded cDNA. Other mechanisms for priming of second strandsynthesis may also occur, including “self-priming” by a hairpin loopformed at the 3′ terminus of the first strand cDNA (Efstratiadis et al.(1976), Cell 7, 279; Higuchi et al. (1976), Proc. Natl, Acad, Sci USA73, 3146; Maniatis et al. (1976), Cell 8, 163; and Rougeon and Mach(1976), Proc. Natl. Acad. Sci. USA 73, 3418; and “non-specific priming”by other DNA molecules in the reaction, i.e. the promoter-primer.

The second strand cDNA synthesis results in the creation of adouble-stranded promoter region. The second strand cDNA includes notonly a sequence of nucleotide residues that comprise a DNA copy of themRNA template, but also additional sequences at its 3′ end which arecomplementary to the promoter-primer used to prime first strand cDNAsynthesis. The double-stranded promoter region serves as a recognitionsite and transcription initiation site for RNA polymerase, which usesthe second strand cDNA as a template for multiple rounds of RNAsynthesis during the next stage of the subject methods.

Depending on the particular protocol, the same or different DNApolymerases may be employed during the cDNA synthesis step. In apreferred embodiment, a single reverse transcriptase, most preferablyMMLV-RT, is used as a source of all the requisite activities necessaryto convert primer-mRNA hybrid to double-stranded cDNA. In anotherpreferred embodiment, the polymerase employed in first strand cDNAsynthesis is different from that which is employed in second strand cDNAsynthesis. Specifically, a reverse transcriptase lacking RNaseH activity(e.g. Superscript II™) is combined with the primer-mRNA hybrid during afirst substep for first strand synthesis. A source of RNaseH activity,such as E. coli RNaseH or MMLV-RT, but most preferably MMLV-RT, is addedduring a second substep to initiate second strand synthesis. In yetother embodiments, the requisite activities are provided by a pluralityof distinct enzymes. The manner is which double-stranded cDNA isproduced from the initial mRNA is not critical to certain embodiments ofthe invention. However, the preferred embodiments use MMLV-RT, or acombination of Superscript II™ and MMLV-RT, or a combination ofSuperscript II™ and E. coli RNaseH, for cDNA synthesis as theseembodiments yield certain desired results. Specifically, in thepreferred embodiments, reaction conditions were chosen so that enzymespresent during the cDNA synthesis do not adversely affect the subsequenttranscription reaction. Potential inhibitors include, but are notlimited to, RNase contaminants of certain enzyme preparations.

The next step of the subject method is the preparation of antisense RNAfrom the double-stranded cDNA prepared in the first step. During thisstep, the double-stranded cDNA produced in the first step is transcribedby RNA polymerase to yield antisense RNA, which is complementary to theinitial mRNA target from which it is amplified. A critical feature ofthe invention is that this second step is carried out in the presence ofreverse transcriptase which is present in the reaction mixture from thefirst step. Thus, the subject methods do not involve a step in which thedouble-stranded cDNA is physically separated from the reversetranscriptase following double-stranded cDNA preparation. Critical tothe subject methods is that the reverse transcriptase that is presentduring the transcription step is rendered inactive. Thus, thetranscription step is carried out in the presence of a reversetranscriptase that is unable to catalyze RNA-dependent DNA polymeraseactivity, at least for the duration of the transcription step. As aresult, the antisense RNA products of the transcription reaction cannotserve as substrates for additional rounds of amplification, and theamplification process cannot proceed exponentially.

The reverse transcriptase present during the transcription step may berendered inactive using any convenient protocol. The transcriptase maybe irreversibly or reversibly rendered inactive. Where the transcriptaseis reversibly rendered inactive, the transcriptase is physically orchemically altered so as to no longer able to catalyze RNA-dependent DNApolymerase activity. The transcriptase may be irreversibly inactivatedby any convenient means. Thus, the reverse transcriptase may be heatinactivated, in which the reaction mixture is subjected to heating to atemperature sufficient to inactivate the reverse transcriptase prior tocommencement of the transcription step. In these embodiments, thetemperature of the reaction mixture and therefore the reversetranscriptase present therein is typically raised to 55° C. to 70° C.for 5 to 60 minutes, usually to about 65° C. for 15 to 20 minutes.Alternatively, reverse transcriptase may irreversibly inactivated byintroducing a reagent into the reaction mixture that chemically altersthe protein so that it no longer has RNA-dependent DNA polymeraseactivity. In yet other embodiments, the reverse transcriptase isreversibly inactivated. In these embodiments, the transcription may becarried out in the presence of an inhibitor of RNA-dependent DNApolymerase activity. Any convenient reverse transcriptase inhibitor maybe employed which is capable of inhibiting RNA-dependent DNA polymeraseactivity a sufficient amount to provide for linear amplification.However, these inhibitors should not adversely affect RNA polymeraseactivity. Reverse transcriptase inhibitors of interest include ddNTPs,such as ddATP, ddCTP, ddGTP or ddTTP, or a combination thereof, thetotal concentration of the inhibitor typically ranges from about 50 μMto 200 μM.

For this transcription step, the presence of the RNA polymerase promoterregion on the double-stranded cDNA is exploited for the production ofantisense RNA. To synthesize the antisense RNA, the double-stranded DNAis contacted with the appropriate RNA polymerase in the presence of thefour ribonucleotides, under conditions sufficient for RNA transcriptionto occur,. where the particular polymerase employed will be chosen basedon the promoter region present in the double-stranded DNA, e.g. T7 RNApolymerase, T3 or SP6 RNA polymerases, E. coli RNA polymerase, and thelike. Suitable conditions for RNA transcription using RNA polymerasesare known in the art, see e.g. Milligan and Uhlenbeck (1989), Methods inEnzymol. 180, 51. As mentioned above, a critical feature of the subjectmethods is that this transcription step is carried out in the presenceof a reverse transcriptase that has been rendered inactive, e.g. by heatinactivation or by the presence of an inhibitor.

Because of the nature of the subject methods, all of the necessarypolymerization reactions, i.e., first strand cDNA synthesis, secondstrand cDNA synthesis and antisense RNA transcription, may be carriedout in the same reaction vessel at the same temperature, such thattemperature cycling is not required. As such, the subject methods areparticularly suited for automation, as the requisite reagents for eachof the above steps need merely be added to the reaction mixture in thereaction vessel, without any complicated separation steps beingperformed, such as phenol/chloroform extraction. A further feature ofthe subject invention is that, despite its simplicity, it yields highamplification extents, where the amplification extents (mass of RNAproduct/mass of RNA target) typically are at least about 50-fold,usually at least about 200-fold and may be as high as 600-fold orhigher. Furthermore, such amplification extents are achieved with lowvariability, e.g. coefficients of variation about the mean amplificationextents that do not exceed about 10%, and usually do not exceed about5%.

The resultant antisense RNA produced by the subject methods finds use ina variety of applications. For example, the resultant antisense RNA canbe used in expression profiling analysis on such platforms as DNAmicroarrays, for construction of “driver” for subtractive hybridizationassays, for cDNA library construction, and the like. Especiallyfacilitated by the subject methods are studies of differential geneexpression in mammalian cells or cell populations. The cells may be fromblood (e.g., white cells, such as T or B cells) or from tissue derivedfrom solid organs, such as brain, spleen, bone, heart, vascular, lung,kidney, liver, pituitary, endocrine glands, lymph node, dispersedprimary cells, tumor cells, or the like. The RNA amplificationtechnology can also be applied to improve methods of detecting andisolating nucleic acid sequences that vary in abundance among differentpopulations using the technique known as subtractive hybridization. Insuch assays, two nucleic acid populations, one sense and the otherantisense, are allowed to mix with one another with one population beingpresent in molar excess (“driver”). Under appropriate conditions, thesequences represented in both populations form hybrids, whereassequences present in only one population remains single-stranded.Thereafter, various well known techniques are used to separate theunhybridized molecules representing differentially expressed sequences.The amplification technology described herein may be used to constructlarge amounts of antisense RNA for use as “driver” in such experiments.

Depending on the particular intended use of the subject antisense RNA,the antisense RNA may be labeled. One way of labeling which may find usein the subject invention is isotopic labeling, in which one or more ofthe nucleotides is labeled with a radioactive label, such as ³²S, ³²P,³H, or the like. Another means of labeling is fluorescent labeling inwhich fluorescently tagged nucleotides, e.g. CTP, are incorporated intothe antisense RNA product during the transcription step. Fluorescentmoieties which may be used to tag nucleotides for producing labeledantisense RNA include: fluorescein, the cyanine dyes, such as Cy3, Cy5,Alexa 542, Bodipy 630/650, and the like. Other labels may also beemployed as are known in the art.

Also provided are kits for use in the subject invention, where such kitsmay comprise containers, each with one or more of the various reagents(typically in concentrated form) utilized in the methods, including, forexample, buffers, the appropriate nucleotide triphosphates (e.g. dATP,dCTP, dGTP, dTTP, ATP, CTP, GTP and UTP), reverse transcriptase, RNApolymerase, and the promoter-primer of the present invention. Alsopresent in kits according to the subject invention is a reversetranscriptase inhibitor, where in many embodiments, the inhibitor is atleast one ddNTP or a combination of ddNTPs, e.g. ddATP and/or ddGTP. Aset of instructions for use of kit components in an mRNA amplificationmethod of the present invention, will also be typically included, wherethe instructions may be associated with a package insert and/or thepackaging of the kit or the components thereof.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL I. Materials and Methods, Example 1

A. Reagents:

cDNA Synthesis Reaction

1. Poly-A RNA. Human HeLa cell poly-A RNA can be purchased fromClontech, P/N 6522-1 (SEQ ID NO:01) DNA T7T18VN (20 μM): (5′) AAT TAATAC GAC TCA CTA TAG GGA GAT TTT TTT TTT TTT TTT TTV N (3′) (V = A/C/G, N= A/C/G/T)

-   3. MMLV Reverse Transcriptase (50 U/μl), Epicentre P/N M4425H-   4. RNAGuard, Pharmacia P/N 27-0815-01-   5. 5× First Strand Buffer: 250 mM Tris-HCl, pH 8.3, 15 mM MgCl₂, 375    mM KCl, Life Technologies P/N 18057-018-   6. 100 mM DTT, supplied with MMLV Reverse Transcriptase, Epicentre-   7. dNTPs (10 mM each), diluted from Pharmacia P/N 2702035-01-   8. Nuclease-free water, Amresco P/N E476    Transcription Reaction-   1. T7 RNA Polymerase, Epicentre P/N TU950K-   2. RNAGuard, Pharmacia P/N 27-0815-01-   3. Inorganic Pyrophosphatase (200 U/ml), diluted from Sigma P/N    1-1891-   4. 5× Transcription Buffer: 0.2 M Tris-HCl, pH 7.5, 50 mM NaCl, 30    mM MgCl₂, 10 mM spermidine, Epicentre P/N BP001-   5. 100 mM DTT, Epicentre P/N BP001-   6. MgCl₂ (200 mM), diluted from Sigma P/N M-1028-   7. NTPs (25 mM ATP, GTP, UTP, 7.5 mM CTP), diluted from Pharmacia    P/N 27-2025-01-   8. Cy3-CTP OR Cy5-CTP (7.0 mM)-   9. Nuclease-free water, Amresco P/N E476-   10. Lithium Chloride (4.0 M), diluted from Sigma P/N L-7026-   11. 70% ethanol, diluted from Amresco E193-   12. TE pH 8.0, Amresco P/N E112    B. Procedure:-   1. Add 100 ng to 600 ng poly-A RNA to reaction tube. Add 1.0 μl DNA    T7T18VN (20 μM) and bring total sample volume to 11.5 μl in    nuclease-free water. Incubate 65 C. for 10 min to denature primer    and template. Quick chill on ice.

2. Mix the following components and maintain on ice. For more than onereaction, multiply by the number of reactions. cDNA Mix Component Volume(μl) 5× First Strand Buffer 4.0 100 mM DTT 2.0 dNTPs (10 mM each) 1.0MMLV-RT (50 U/μl) 1.0 RNAGuard (36 U/μl) 0.5 Volume of cDNA Mix 8.5

-   3. Aliquot 8.5 μl of cDNA Mix into each sample tube.

 Incubate cDNA synthesis reaction at 40 C. for 120 min. Composition ofcDNA Synthesis Reaction Component Final concentration or amount poly-ARNA 200 ng DNA T7T18VN 1 μM Tris-HCl, pH 8.3 50 mM MgCl₂ 3.0 mM KCl 75mM DTT 10 mM dNTPs 0.5 mM each MMLV-RT 50 U RNAGuard 18 U Total reactionvolume 20 μl

-   4. Incubate reaction tubes at 65 C. for 15 min.-    Move reaction tubes to ice. Store reactions tubes on ice for 5 min.

5. Immediately before use, mix the following components at roomtemperature. For more than one reaction, multiply by the number ofreactions. Transcription Mix Component Volume (μl) Nuclease-free water20.8 5× Transcription Buffer 16 100 mM DTT 6.0 NTPs (25 mM A, G, U, 7.5mM CTP) 8.0 Cy3-CTP (7.0 mM) OR Cy5-CTP (7.0 mM) 4.0 200 mM MgCl₂ 3.3RNAGuard (36 U/μl) 0.5 Inorganic Pyrophosphatase (200 U/ml) 0.6 T7 RNApolymerase (2500 U/μl) 0.8 Volume of Transcription Mix 60

-   6. Aliquot 60 μl of Transcription Mix into each sample tube.

 Incubate transcription reactions at 40 C. for 60 min. Composition ofTranscription Reaction Component Final concentration or amountDouble-strand cDNA Approximately 400 ng Tris-HCl, pH 7.5 52 mM MgCl₂ 15mM KCl 19 mM NaCl 10 mM Spermidine 2 mM DTT 10 mM ATP, GTP, UTP 2.5 mMeach CTP 0.75 mM Cy3-CTP OR Cy5-CTP 350 μM T7 RNA polymerase 2000 URNAGuard 18 U Inorganic pyrophosphatase 0.12 U Total reaction volume 80μl

-   7. Add 20 μl 50 mM EDTA to each reaction tube to stop the reaction.    Alternatively, if quantitation of the amplified RNA product is    desired, purifiy the RNA from unincorporated nucleotides by    precipitation of the antisense RNA products in 2.0 M LiCl. Measure    the RNA concentration by absorbance at OD₂₆₀ using the conversion: 1    OD₂₆₀=40 μl g/ml RNA. Measure concentration of Cy3-CTP or Cy5-CTP by    absorbance at OD₅₅₂ (ε=150 (1/mMcm)) or (ε=250 (1/mMcm)),    respectively.

II. Materials and Methods, Example 2

A. Reagents:

First Strand cDNA Synthesis

1. Poly-A RNA. Human HeLa cell poly-A RNA can be purchased fromClontech, P/N 6522-1 (SEQ ID NO:01) DNA T7T18VN (20 μu): (5′) AAT TAATAC GAC TCA CTA TAG GGA GAT TTT TTT TTT TTT TTT TTV N (3′) (V = A/C/G, N= A/C/G/T)

-   3. Superscript II Reverse Transcriptase, Life Technologies P/N    18064-014-   4. RNAGuard, Pharmacia P/N 27-0815-01-   5. 5× First Strand Buffer*: 250 mM Tris-HCl, pH 8.3, 15 mM MgCl₂,    375 mM KCl    * supplied with Superscript II Reverse Transcriptase, Life    Technologies-   6. 100 mM DTT, supplied with Superscript II Reverse Transcriptase,    Life Technologies-   7. dNTPs (10 mM each), diluted from Pharmacia P/N 2702035-01-   8. Nuclease-free water, Amresco P/N E476    Second Strand cDNA Synthesis-   1. MMLV Reverse Transcriptase, Epicentre P/N M4425H-   2. 5× First Strand Buffer*: 250 mM Tris-HCl, pH 8.3, 15 mM MgCl₂,    375 mM KCl, supplied with Superscript II Reverse Transcriptase, Life    Technologies-   3. dNTPs (10 mM each), diluted from Pharmacia P/N 2702035-01-   4. Nuclease-free water, Amresco P/N E476    Transcription Reaction-   1. T7 RNA Polymerase, Epicentre P/N TU950K-   2. RNAGuard, Pharmacia P/N 27-0815-01-   3. Inorganic Pyrophosphatase (200 U/ml), diluted from Sigma P/N    I-1898-   4. 5× Transcription Buffer: 0.2 M Tris-HCl, pH 7.5, 50 mM NaCl, 30    mM MgCl₂, 10 mM spermidine, Epicentre P/N BP001-   5. 100 mM DTT, Epicentre P/N BP001-   6. MgCl₂ (400 mM), diluted from Sigma P/N M-1028-   7. NTPs (25 mM ATP, GTP, UTP, 7.5 mM CTP), diluted from Pharmacia    P/N 27-2025-01-   8. Cy3-CTP OR Cy5-CTP (7.0 mM)-   9. ddATP (5 mM), Pharmacia P/N 27-2057-01-   10. ddGTP (5 mM), Pharmacia P/N 27-2075-01-   11. Nuclease-free water, Amresco P/N E476-   12. Lithium Chloride (4.0 M), diluted from Sigma P/N L-7026-   13. 70% ethanol, diluted from Amresco E193-   14. TE pH 8.0, Amresco P/N E112    B. Procedure:-   1. Add 100 ng to 600 ng poly-A RNA to reaction tube. Add 1.0 μl DNA    T7T18VN (20 μM) and bring total sample volume to 11.5 μl in    nuclease-free water. Incubate 70 C. for 10 min to denature primer    and template. Quick chill on ice.

2. Mix the following components and maintain on ice. For more than onereaction, multiply by the number of reactions. Master Mix A ComponentVolume (μl) 5× First Strand Buffer 4.0 100 mM DTT 2.0 dNTPs (10 mM each)1.0 Superscript II RT (200 U/μl) 1.0 RNAGuard (36 U/μl) 0.5 Volume ofMaster Mix A 8.5

3. Aliquot 8.5 μl of the master mix A into each sample tube. Incubatefirst strand synthesis reaction at 40 C. for 60 min. Composition ofFirst Strand Synthesis Reaction Component Final concentration or amountpoly-A RNA 200 ng DNA T7T18VN 1 μM Tris-HCl, pH 8.3 50 mM MgCl₂ 3 mM KCl75 mM DTT 10 mM dNTPs 0.5 mM each Superscript II RT 200 U RNAGuard 18 UTotal reaction volume 20 μl

4. Mix the following components and maintain on ice. For more than onereaction, multiply by the number of reactions. Master Mix B ComponentVolume (μl) 5× First Strand Buffer 4.0 dNTPs (10 mM each) 1.0 MMLV-RT(50 U/μl) 1.0 Nuclease-free water 13 Volume of master mix B 20

5. Aliquot 20 μl of the Master Mix B into each sample tube. Incubatesecond strand synthesis reaction at 40 C. for 60 min. Composition ofSecond Strand Synthesis Reaction Component Final concentration or amountcDNA Approximately 200 ng Tris-HCl, pH 8.3 50 mM MgCl₂ 3 mM KCl 75 mMDTT 5 mM dNTPs 0.5 mM each MMLV-RT 50 U Total reaction volume 40 μl

6. Immediately before use, mix the following components at roomtemperature. For more than one reaction, multiply by the number ofreactions. Master Mix C Component Volume (μl) Nuclease-free water 0.8 5×Transcription Buffer 16 100 mM DTT 6.0 NTPs (25 mM A, G, U, 7.5 mM CTP)8.0 Cy3-CTP OR Cy5-CTP (7.0 mM) 4.0 400 mM MgCl₂ 1.7 ddATP (5.0 mM) 0.8ddGTP (5.0 mM) 0.8 RNAGuard (36 U/μl) 0.5 Inorganic Pyrophosphatase (200U/μl) 0.6 T7 RNA polymerase (2500 U/μl) 0.8 Volume of Master Mix C 40

7. Aliquot 40 μl of the master mix C into each sample tube. Incubatetranscription reactions at 40 C. for 60 min. Composition ofTranscription Reaction Component Final concentration or amountDouble-strand cDNA Approximately 400 ng Tris-HCl, pH 8.1 65 mM MgCl₂ 16mM KCl 37.5 mM NaCl 10 mM Spermidine 2 mM DTT 10 mM ATP, GTP, UTP 2.5 mMeach CTP 0.75 mM Cy3-CTP OR Cy5-CTP 350 μM ddATP, ddGTP 50 μM each T7RNA polymerase 2000 U RNAGuard 18 U Inorganic pyrophosphatase 0.12 UTotal reaction volume 80 μl

-   8. Add 20 μl 50 mM EDTA to each reaction tube to stop the reaction.    Alternatively, if quantitation of the amplified RNA product is    desired, purifiy the RNA from unincorporated nucleotides by    precipitation of the antisense RNA products in 2.0 M LiCl. Measure    the RNA concentration by absorbance at OD₂₆₀ using the conversion: 1    OD₂₆₀=40μg/ml RNA. Measure concentration of Cy3-CTP or Cy5-CTP by    absorbance at OD₅₅₂ (ε=150 (1/mMcm)) or (ε=250 (1/mMcm)),    respectively.

III. Results

The above protocols provide two variations of the subject method for theamplification of heterogeneous mRNA populations. Both embodiments of theprocedure perform essentially equivalently and yield amplificationextents (mass of antisense RNA product/mass of mRNA target) up to600-fold. The products of the amplification reaction are antisense RNAmolecules with a size distribution resembling that of the cellular mRNAtargets. The antisense RNA products accumulate linearly with respect totime, and thus bias in the relative amounts of specific RNA productsassociated with exponential amplification is not likely to occur. Infact, the pattern of hybridization intensities on cDNA arrays usingfluorescently labeled amplified RNA is very similar to those obtainedusing labeled cDNA target.

FIG. 1 shows that the amplification method described herein showssignificantly higher performance than that described by U.S. Pat. No.5,716,785. The protocol describing a modification of the methoddescribed in this patent that was used for this analysis is found inAppendix A, infra. Amplification extents (micrograms of RNAproduct/micrograms RNA target) using the amplification method describedin this disclosure were significantly higher (average=300) with lowervariability (coefficient of variation (CV)=7.0%) than those using themethod described by Van Gelder et al. (average=69, CV=35%).

The above results and discussion demonstrate that novel and improvedmethods of producing linearly amplified amounts of antisense RNA from aninitial mRNA source are provided. The subject methods provide for animprovement over prior methods of producing antisense RNA in that thestep of separating the double-stranded cDNA from the reversetranscriptase used for its preparation is not required. Instead, all ofthe steps for linearly amplifying mRNA into antisense RNA may by carriedout in a single reaction mixture without performing a separation step.As such, the subject methods are amenable to automation, making themparticularly attractive for high throughput applications. Furthermore,linear amplification extents of 600-fold and labeling efficiencies of 8%can be achieved using the subject methods. Finally, all of the benefitsof linear amplification are achieved with the subject methods, such asthe production of unbiased antisense RNA libraries from heterogeneousmRNA mixtures. As such, the subject methods represent a significantcontribution to the art.

All publications and patent application cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

APPENDIX A

Antisense RNA Amplification Using a Modification of the Method Describedin Van Gelder et al. U.S. Pat. No. 5,716,785

A. Reagents:

First Strand cDNA Synthesis

-   1. Poly-A RNA. Human HeLa Cell poly-A RNA can be purchased from    Clontech, P/N 6522-1-   2. DNA T7T18VN (20 μM): (5′) AAT TAA TAC GAC TCA CTA TAG GGA GAT TTT    TTT TTT TTT TTT TTV N (3′) (V=A/C/G, N=A/C/G/T)-   3. Superscript II Reverse Transcriptase, Life Technologies P/N    18064-014-   4. RNAGuard, Pharmacia P/N 27-0815-01-   5. 5× First Strand Buffer*: 250 mM Tris-HCl, pH 8.3, 15 mM MgCl₂,    375 mM KCl    * supplied with Superscript II Reverse Transcriptase, Life    Technologies-   6. 100 mM DTT*    * supplied with Superscript II Reverse Transcriptase, Life    Technologies-   7. dNTPs (10 mM each), diluted from Pharmacia P/N 2702035-01-   8. Nuclease-free water, Amresco P/N E476    Second Strand cDNA Synthesis-   12. E. coli DNA polymerase I, Life Technologies P/N 18010-017-   12. E. coli RNaseH, Life Technologies P/N 18021-014-   12. E. coli DNA Ligase, Life Technologies P/N 18052-019-   12. T4 DNA polymerase, Epicentre P/N D0602H-   12. 5× Second Strand Buffer: 100 mM Tris-HCl, pH 6.9, 23 mM MgCl₂,    450 mM KCl, 0.75 mM β-NAD+, 50 mM (NH₄)₂SO₄, Life Technologies P/N    10812014-   12. dNTPs (10 mM each), diluted from Pharmacia P/N 2702035-01-   12. Nuclease-free water, Amresco P/N E476-   12. EDTA 0.5 M, Amresco P/N E522 (or equivalent)-   12. Phenol:chloroform:isoamyl alcohol (25:24:1), Amresco P/N 0883    (or equivalent)-   12. Ammonium Acetate 7.5 M, Sigma P/N A2706-   12. 100% ethanol, Amresco P/N E193-   12. 70% ethanol, diluted from Amresco P/N E193    Transcription Reaction-   1. T7 RNA Polymerase, Epicentre P/N TU950K-   2. RNAGuard, Pharmacia P/N 27-0815-01-   3. Inorganic Pyrophosphatase (200 U/ml)-   4. 5× Transcription Buffer: 0.2 M Tris-HCl, pH 7.5, 50 mM NaCl, 30    mM MgCl₂, 10 mM spermidine, Epicentre P/N BP001-   5. 100 mM DTT, Epicentre P/N BP001-   6. MgCl₂ (400 mM), diluted from Sigma P/N M-1028-   7. NTPs (25 mM each), diluted from Pharmacia P/N 27-2025-01-   8. Nuclease-free water, Amresco P/N E476-   9. Lithium Chloride (4.0 M), diluted from Sigma P/N L-7026-   10. 70% ethanol, diluted from Amresco P/N E193-   11. TE pH 8.0, Amresco P/N E112    B. Procedure:-   1. Add 100 ng to 600 ng poly-A RNA to reaction tube. Add 1.0 μl DNA    T7T18VN (20 μM) and bring total sample volume to 11.5 μl in    nuclease-free water. Incubate 70 C. for 10 min to denature primer    and template. Quick chill on ice.

2. Mix the following components and maintain on ice. For more than onereaction, multiply by the number of reactions. Master Mix A ComponentVolume (μl) 5× First Strand Buffer 4.0 100 mM DTT 2.0 dNTPs (10 mM each)1.0 Superscript II RT (200 U/μl) 1.0 RNAGuard (36 U/μl) 0.5 Volume ofMaster Mix A 8.5

3. Aliquot 8.5 μl of the master mix A into each sample tube. Incubatefirst strand synthesis reaction at 37 C. for 60 min. Composition ofFirst Strand Synthesis Reaction Component Final concentration or amountpoly-A RNA 600 ng DNA T7T18VN 1 μM Tris-HCl, pH 8.3 50 mM MgCl₂ 3 mM KCl75 mM DTT 10 mM dNTPs 0.5 mM each Superscript II RT 200 U RNAGuard 18 UTotal reaction volume 20 μl

-   4. Mix the following components and maintain on ice. For more than    one reaction, multiply by the number of reactions.

 Note: If the activities of the enzymes are different than indicatedbelow, add the appropriate volume to give the Units per reactionindicated below. Master Mix B Component Volume (μl) Units Nuclease-freewater 91 5× Second Strand Buffer 30 dNTPs (10 mM each) 3.0 E. coli DNALigase (10 U/μl) 1.0 10 E. coli DNA polymerase I (10 U/μl) 4.0 40 E.coli RNaseH (2 U/μl) 1.0 2 Volume of master mix B 130

-   5. On ice, aliquot 130 μl of the Master Mix B into each sample.    Incubate second strand synthesis reaction at 16 C. for 120 min.

 Note: Second strand synthesis reactions are incubated at 16 C. toinhibit strand displacement by DNA polymerase I. Do not let thetemperature rise above 16 C. Composition of Second Strand SynthesisReaction Component Final concentration or amount Single-strand cDNAApproximately 600 ng Tris-HCl, pH 7.5 25 mM MgCl₂ 5 mM KC1 100 mM β-NAD⁺0.15 mM 10 mM (NH4)₂SO₄ 10 mM DTT 1.2 mM dNTPs 0.25 mM each E. coli DNALigase 10 U E. coli DNA Polymerase I 40 U E. coli RNaseH 2 U Totalreaction volume 150 μl

-   6. Add 2 μl T4 DNA polymerase (10 U) to each sample tube. Incubate    16 C. for 5 min.-   7. Add 10 μl of 0.5 M EDTA to each sample tube.-   8. Add 150 μl of phenol:chloroform:isoamyl alcohol (25:24:1). Vortex    thoroughly and spin 5 min at room temperature in microcentrifuge.    Carefully remove 140 μl of the upper, aqueous phase and transfer to    fresh reaction tube.-   9. Add 70 μl 7.5 M NH₄OAc and 0.5 ml 100% ethanol to each sample    tube. Mix and spin 20 min at 4 C. in microcentrifuge. Rinse each    sample pellet in 70% ethanol. Dry pellet briefly at room    temperature. Resuspend each sample pellet in 40 μl nuclease-free    water.

10. Immediately before use, mix the following components at roomtemperature. For more than one reaction, multiply by the number ofreactions. Master Mix C Component Volume (μl) Nuclease-free water 4.1 5×Transcription Buffer 16 100 mM DTT 8.0 NTPs (25 mM each) 8.0 400 mMMgCl₂ 2.0 RNAGuard (36 U/μl) 0.5 Inorganic Pyrophosphatase (200 U/μl)0.6 T7 RNA polymerase (2500 U/μl) 0.8 Volume of Master Mix C 40

11. Aliquot 40 μl of the master mix C into each sample tube. Incubatetranscription reactions at 37 C. for 60 min. Composition ofTranscription Reaction Component Final concentration or amountDouble-strand cDNA Approximately 1200 ng Tris-HCl, pH 7.5 50 mM MgCl₂ 16mM NaCl 10 mM Spermidine 2 mM DTT 10 mM NTPs 2.5 mM each T7 RNApolymerase 2000 U RNAGuard 18 U Inorganic pyrophosphatase 0.12 U Totalreaction volume 80 μl12. Add 20 μl 50 mM EDTA to each reaction tube to stop the reaction.Alternatively, if quantitation of the amplified RNA product is desired,purifiy the RNA from unincorporated nucleotides by precipitation of theantisense RNA products in 2.0 M LiCl. Measure the RNA concentration byabsorbance at OD₂₆₀ using the conversion: 1 OD₂₆₀=40 μg/ml RNA.

1-31. (canceled)
 32. A method for producing RNA, comprising: (a)converting an RNA molecule to double-stranded cDNA, wherein one terminusof the double-stranded cDNA comprises an RNA polymerase promoter region;and (b) transcribing the double-stranded cDNA with an RNA polymerase andribonucleotides in the presence of a reverse transcriptase that isincapable of RNA-dependent DNA polymerase activity during thetranscribing step.
 33. The method according to claim 32, wherein themethod further comprises inactivating the reverse transcriptase prior tothe transcribing step.
 34. The method according to claim 33, wherein theinactivation is accomplished by heating the reaction mixture.
 35. Themethod according to claim 32, wherein the method further comprisesinhibiting the reverse transcriptase with an inhibitor during thetranscribing step.
 36. The method according to claim 35, wherein theinhibitor is at least one ddNTP.
 37. The method according to claim 32,wherein the converting step comprises a single cDNA synthesis step,wherein the same polymerase is employed for the synthesis of first andsecond cDNA strands.
 38. The method according to claim 32, wherein theconverting step comprises a first strand cDNA synthesis step and asecond strand cDNA synthesis step.
 39. The method according to claim 38,wherein a first polymerase is employed for synthesis of the first strandcDNA, which is lacking RNaseH activity.
 40. The method according toclaim 32, wherein the converting step employs a promoter-primercomprising an RNA binding site for the RNA molecule linked to a promotersequence.
 41. A method for producing RNA comprising: (a) converting anRNA molecule to cDNA with a promoter-primer comprising a binding sitefor the RNA molecule linked to an RNA polymerase promoter sequence; and(b) transcribing the cDNA with an RNA polymerase and ribonucleotides inthe presence of a reverse transcriptase that has been renderedineffective for RNA-dependent DNA polymerase activity prior to thetranscribing step.
 42. The method according to claim 41, wherein themethod further comprises inactivating the reverse transcriptase prior tothe transcribing step.
 43. The method according to claim 42, wherein theinactivation is accomplished by heating the reaction mixture.
 44. Themethod according to claim 41, wherein the method further comprisesinhibiting the reverse transcriptase with an inhibitor during thetranscribing step.
 45. The method according to claim 44, wherein theinhibitor is at least one ddNTP.
 46. A method for producing RNAcomprising: (a) converting an RNA molecule to double-stranded cDNA by:(i) contacting the RNA molecule with a promoter-primer under conditionswherein the RNA molecule forms a complex with the promoter-primer,wherein the promoter-primer comprises a binding site for the RNAmolecule linked to a RNA promoter sequence; and (ii) converting thecomplex to double-stranded cDNA using a combination of RNA-dependent DNApolymerase activity, RNaseH activity and DNA-dependent DNA polymeraseactivity; and (b) transcribing the double-stranded cDNA with an RNApolymerase and ribonucleotides in the presence of a reversetranscriptase that is incapable of RNA-dependent DNA polymerase activityduring the transcribing step.
 47. The method according to claim 46,wherein the RNA-dependent DNA polymerase activity, RNaseH activity andDNA-dependent DNA polymerase activity are contributed by a singlepolymerase.
 48. The method according to claim 47, wherein the polymeraseis the reverse transcriptase of Moloney Murine leukemia virus (MMLV-RT).49. The method according to claim 47, wherein the polymerase is thereverse transcriptase of avian myeloblastosis virus (AMV-RT).
 50. Themethod according to claim 46, wherein the RNA-dependent DNA polymeraseactivity is inhibited with ddNTPs.
 51. A method for producing RNAcomprising: (a) contacting an RNA molecule with a promoter-primer in thepresence of a first polymerase having RNA-dependent DNA polymeraseactivity and lacking RNaseH activity under conditions sufficient forfirst strand cDNA synthesis to occur to produce a hybrid of the RNAmolecule and a first strand cDNA, wherein the promoter-primer comprisesan binding site for the RNA molecule linked to a RNA polymerase promotersequence; (b) contacting the hybrid with an enzyme having RNaseHactivity under conditions sufficient to convert the complex to adouble-stranded cDNA molecule; and (c) transcribing the double-strandedcDNA with an RNA polymerase and ribonucleotides in the presence ofddNTPs.
 52. The method according to claim 51, wherein the enzymecatalyzing RNaseH activity is the reverse transcriptase of MoloneyMurine leukemia virus (MMLV-RT).
 53. The method according to claim 51,wherein the enzyme catalyzing RNaseH activity is the RNaseH ofEscherichia coli (E. coli RNaseH).
 54. The method according to claim 51,wherein the RNA polymerase promoter is the T7 promoter and the RNApolymerase is T7 RNA polymerase.
 55. The method according to claim 51,wherein the RNA polymerase promoter is the T3 promoter and the RNApolymerase is T3 RNA polymerase.
 56. The method according to claim 51,wherein the ddNTPs are selected from the group consisting of: ddATP andddGTP.